Expression vectors encoding Bacillus subtilis disulfide bond isomerase and methods of secreting proteins in gram-positive microorganisms using the same

ABSTRACT

The present invention relates to nucleic acid sequences and amino acid sequence for  Bacillus subtills  disulfide bond isomerases, Dsb1 and Dsb2 and methods for increasing the secretion of heterologous and homologous proteins in gram-positive microorganisms.

FIELD OF THE INVENTION

The present invention generally relates to expression of proteins ingram-positive microorganisms and specifically to gram positivemicroorganism disulfide bond isomerases. The present invention providesexpression vectors, methods and systems for the production of proteinsin gram-positive microorganisms.

BACKGROUND OF THE INVENTION

Gram-positive microorganisms, such as members of the group Bacillus,have been used for large-scale industrial fermentation due, in part, totheir ability to secrete their fermentation products into the culturemedia. In gram-positive bacteria, secreted proteins are exported acrossa cell membrane and a cell wall, and then are subsequently released intothe external media usually obtaining their native conformation.

The folding of polypeptide chains depends upon the assistance ofmolecular chaperones and folding catalysts, such as disulfide bondisomerases, which accelerate specific steps in folding. (Hartl et al.,1995, Current Opinion in Structural Biology, 5:92-102). Disulfide bondisomerases can covalently modify proteins by catalyzing specificisomerization steps that may limit the folding rate of some proteins(PCT/US93/09426).

Disulfide bond isomerase catalyzes thiol/disulfide interchange reactionsand promotes disulfide formation, isomerization or reduction, therebyfacilitating the formation of the correct disulfide pairings, and mayhave a more general role in the prevention and premature misfolding ofnewly translocated chains. Disulfide bond isomerase interacts directlywith newly synthesized secretory proteins and is required for thefolding of nascent polypeptides in the endoplasmic reticulum (ER) ofeukaryotic cells.

In spite of advances in understanding portions of the protein secretionmachinery in procaryotic cells, the complete mechanism of proteinsecretion and the mechanisms associated with correct folding of secretedproteins, especially for gram-positive microorganisms has yet to befully elucidated.

SUMMARY OF THE INVENTION

The capacity of the secretion machinery of a Gram-positive microorganismmay become a limiting factor or bottleneck to the secretion of properlyfolded proteins or polypeptides and the production of proteins havingthe correct conformation in secreted form, in particular when theproteins are recombinantly introduced and overexpressed by the hostcell. The present invention provides a means for alleviating that bottleneck.

The present invention is based, in part, upon the discovery of aBacillus subtilis disulfide bond isomerases, Dsb1 and Dsb2, identifiedin heretofore uncharacterised translated genomic DNA by their overallamino acid homology with an E. coli disulfide bond isomerase. For E.coli disulfide bond isomerase having accession number P30018 and IDnumber DSBB_(—) ECOLI, it was noted that cysteine residues at 41, 44,104 and 130 were essential for activity. Those residues are conserved inB. subtilis Dsb1 and Dsb2 and noted in FIGS. 2, 3 and 4. The presentinvention is also based upon the overall structural homology that Dsb1and Dsb2 have with each other. The present invention is also based inpart on the presence in of potential transmembrane spanning regions inDsb1 and Dsb2.

The present invention provides isolated nucleic acid and amino acidsequences for B. subtilis Dsb1 and Dsb2. The amino acid sequence for B.subtilis Dsb1 and Dsb2 are shown in FIGS. 1 and 7, respectfully. Thenucleic acid sequences encoding B. subtilis Dsb1 and Dsb2 are thepolynucleotide sequences shown in FIGS. 1 and 7, respectfully.

The present invention also provides improved methods for secretingcorrectly folded proteins from gram-positive microorganisms.Accordingly, the present invention provides an improved method forsecreting a protein in a gram-positive microorganism comprising thesteps of obtaining a gram-positive microorganism host cell comprisingnucleic acid encoding Dsb1 and/or Dsb2 wherein said nucleic acid isunder the control of expression signals capable of expressing saiddisulfide bond isomerase in a gram-positive microorganism saidmicroorganism further comprising nucleic acid encoding said protein; andculturing said microorganism under conditions suitable for expression ofsaid disulfide bond isomerase and expression and secretion of saidprotein. In one embodiment of the present invention, the protein ishomologous or naturally occurring in the gram-positive microorganism. Inanother embodiment of the present invention, the protein is heterologousto the gram-positive microorganism.

The present invention provides expression vectors and host cellscomprising isolated nucleic acid encoding a gram-positive Dsb1 and/orDsb2. In one embodiment of the present invention, the host cellcomprising nucleic acid encoding Dsb1 or Dsb2 is genetically engineeredto produce a desired protein, such as an enzyme, growth factor orhormone. In yet another embodiment of the present invention, the enzymeis selected from the group consisting of proteases, carbohydrasesincluding amylases, cellulases, xylanases, reductases and lipases;isomerases such as racemases, epimerases, tautomerases, or mutases;transferases, kinases and phophatases acylases, amidases, esterases,oxidases. In a further embodiment the expression of the disulfide bondisomerases Dsb1 and/or Dsb2 is coordinated with the expression of othercomponents of the secretion machinery. Preferably other components ofthe secretion machinery, i.e., translocase, SecA, SecY, SecE and/orother secretion factors known to those of skill in the art are modulatedin expression at an optimal ratio to Dsb1 and Dsb2. For example, it maybe desired to overexpress multiple secretion factors in addition to Dsb1or Dsb2 for optimum enhancement of the secretion patterns.

The present invention also provides a method of identifying homologousnon Bacillus subtilis Dsb1 and Dsb2 that comprises hybridizing part orall of dsb1 and dsb2 nucleic acid shown in the Figures with nucleic acidderived from gram-positive microorganisms. In one embodiment, thenucleic acid is of genomic origin. In another embodiment, the nucleicacid is a cDNA. The present invention encompasses novel gram-positivemicroorganism disulfide bond isomerases identified by this method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleic acid (SEQ ID NO: 1) and deduced amino acidsequence for Dsb1 (SEQ ID NO: 2).

FIG. 2 shows an amino acid alignment of E. coli disulfide bond isomerase(SEQ ID NO: 3) with the deduced amino acid sequence for Dsb1 (YOLK) (SEQID NO: 2) The cysteine residues equivalent to E. coli cysteine residuesat 41, 44, 104 and 130 found to be essential for activity of the E. colidisulfide bond isomerase are marked in FIGS. 2, 3 and 4.

FIG. 3 shows an amino acid alignment of E. coli DSB (SEQ ID NO: 3) vsDsb2 (YVGU) (SEQ ID NO: 5).

FIG. 4 shows an amino acid alignment of Dsb1 (YOLK) (SEQ ID NO: 2) vsDsb2 (YVGU) (SEQ ID NO: 5).

FIG. 5 shows a hydrophilicity plot for Dsb1 (YOLK).

FIG. 6 shows a hydrophilicity plot of Dsb 2 (YVGU).

FIG. 7 shows the nucleic acid (SEQ ID NO: 4) and deduced amino acidsequence for Dsb2 (SEQ ID NO: 5).

DETAILED DESCRIPTION

Definitions

As used herein, the genus Bacillus includes all members known to thoseof skill in the art, including but not limited to B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus and B. thuringiensis.

The present invention encompasses novel Dsb1 and Dsb2 disulfide bondisomerases obtainable from any gram positive organism. In a preferredembodiment, the gram-positive organism is Bacillus. In another preferredembodiment, the gram-positive organism if from B. subtilis. As usedherein, the phrase, “B. subtilis Dsb1” or “B. subtilis disulfide bondisomerase 1” refers to the amino acid sequence shown in FIG. 1. As usedherein, the phrase, “B. subtilis Dsb2” or “B. subtilis disulfide bondisomerase 2” refers to the amino acid sequence shown in FIG. 7. Thepresent invention encompasses amino acid variants of B. subtilis Dsb1and Dsb2 disclosed in FIGS. 1 and 7, respectively, that are able tomodulate protein folding and/or secretion alone or in combination withother molecular factors, such as secretion factors.

As used herein, “nucleic acid” refers to a nucleotide or polynucleotidesequence, and fragments or portions thereof, and to DNA or RNA ofgenomic or synthetic origin which may be double-stranded orsingle-stranded, whether representing the sense or antisense strand.

As used herein “amino acid” refers to peptide or protein sequences orportions thereof. A “B. subtilis polynucleotide homolog” or“polynucleotide homolog” as used herein refers to a polynucleotide thathas at least 80%, at least 90% and at least 95% identity to B. subtilisDsb1 or Dsb2 or which is capable of hybridizing to B. subtilis Dsb1 orDsb2 under conditions of high stringency and which encodes an amino acidsequence that is able to modulate secretion of the gram-positivemicroorganism from which it is derived. Modulate as used herein refersto the ability of a disulfide bond isomerase to alter the secretionmachinery such that secretion of proteins is altered.

The terms “isolated” or “purified” as used herein refer to a nucleicacid or amino acid that is removed from at least one component withwhich it is naturally associated.

As used herein, the term “heterologous protein” refers to a protein orpolypeptide that does not naturally occur in a gram-positive host cell.Examples of heterologous proteins include enzymes such as hydrolasesincluding proteases, cellulases, amylases, other carbohydrases,reductases and lipases; isomerases such as racemases, epimerases,tautomerases, or mutases; transferases, kinases and phophatases. Theheterologous gene may encode therapeutically significant proteins orpeptides, such as growth factors, cytokines, ligands, receptors andinhibitors, as well as vaccines and antibodies. The gene may encodecommercially important industrial proteins or peptides, such asproteases, carbohydrases such as amylases and glucoamylases, cellulases,oxidases and lipases. The gene of interest may be a naturally occurringgene, a mutated gene or a synthetic gene.

The term “homologous protein” refers to a protein or polypeptide nativeor naturally occurring in a gram-positive host cell. The inventionincludes host cells producing the homologous protein via recombinant DNAtechnology. The present invention encompasses a gram-positive host cellhaving a deletion or interruption of the nucleic acid encoding thenaturally occurring homologous protein, such as a protease, and havingnucleic acid encoding the homologous protein, or a variant thereof,re-introduced in a recombinant form. In another embodiment, the hostcell produces the homologous protein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides novel gram-positive microorganismdisulfide bond isomerases Dsb1 and Dsb2 and methods that can be used ingram-positive microorganisms to ameliorate the bottleneck to secretionof proteins in their native or naturally occurring conformation, inparticular when the proteins are recombinantly introduced andoverexpressed by the host cell. The present invention provides thedisulfide bond isomerase Dsb1 and Dsb2 derived from Bacillus subtilis.

I. Dsb1 and Dsb2 Nucleic Acid and Amino Acid Sequences Nucleic Acid

The Dsb1 and Dsb2 polynucleotides having the sequence as shown in FIGS.1 and 7, respectively, encode the Bacillus subtilis disulfide bondisomerase Dsb1 and Dsb2. The Bacillus subtilis Dsb1 and Dsb2 wereidentified via a FASTA search of Bacillus subtilis uncharacterizedtranslated genomic sequences using an E. coli disulfide bond isomerase.The present invention provides gram-positive Dsb1 and Dsb2polynucleotides which may be used alone or together with factors in agram-positive microorganism for the purpose of increasing the secretionof desired heterologous or homologous proteins or polypeptides in theirnative or naturally occurring conformation.

The present invention encompasses novel B. subtilis Dsb1 and Dsb2polynucleotide homologs encoding gram-positive microorganism Dsb1 andDsb2 which have at least 80%, or at least 90% or at least 95% identityto B. subtilis Dsb1 and Dsb2 as long as the homolog encodes a proteinthat is able to function by modulating secretion in a gram-positivemicroorganism. As will be understood by the skilled artisan, due to thedegeneracy of the genetic code, a variety of polynucleotides, i.e., Dsb1and Dsb2 polynucleotide variants, can encode the Bacillus subtilisdisulfide bond isomerase s Dsb1 and Dsb2. The present inventionencompasses all such polynucleotides.

Gram-positive microorganism polynucleotide homologs of B. subtilis Dsb1and Dsb2 can be identified through nucleic acid hybridization ofgram-positive microorganism nucleic acid of either genomic of cDNAorigin. The polynucleotide homolog sequence can be detected by DNA-DNAor DNA-RNA hybridization or amplification using B. subtilis Dsb1 andDsb2 probes, portions or fragments disclosed in the nucleotide sequenceshown in the Figures. Accordingly, the present invention provides amethod for the detection of gram positive microorganism Dsb1 and Dsb2polynucleotide homologs which comprises hybridizing a nucleic acidsample with part or all of a nucleic acid sequence from B. subtilis Dsb1and Dsb2.

Also included within the scope of the present invention aregram-positive microorganism Dsb1 and Dsb2 polynucleotide homologs thatare capable of hybridizing to part or all of B. subtilis Dsb1 and Dsb2under conditions of intermediate to maximal stringency. Hybridizationconditions are based on the melting temperature (Tm) of the nucleic acidbinding complex, as taught in Berger and Kimmel (1987, Guide toMolecular Cloning Techniques, Methods in Enzymology, Vol 152, AcademicPress, San Diego Calif.) incorporated herein by reference, and confer adefined “stringency” as explained below.

“Maximum stringency” typically occurs at about Tm-5° C. (5° C. below theTm of the probe); “high stringency” at about 5° C. to 10° C. below Tm;“intermediate stringency” at about 10° C. to 20° C. below Tm; and “lowstringency” at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical polynucleotide sequences while anintermediate or low stringency hybridization can be used to identify ordetect polynucleotide sequence homologs.

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” (Coombs J (1994) Dictionary of Biotechnology, StocktonPress, New York N.Y.).

The process of amplification as carried out in polymerase chain reaction(PCR) technologies is described in Dieffenbach CW and GS Dveksler (1995,PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, PlainviewN.Y.). A nucleic acid sequence of at least about 10 nucleotides and asmany as about 60 nucleotides from the Dsb1 and Dsb2 nucleotide sequenceof FIG. 1, preferably about 12 to 30 nucleotides, and more preferablyabout 20-25 nucleotides can be used as a probe or PCR primer.

Amino Acid Sequences

The B. subtilis Dsb1 and Dsb2 polynucleotide as shown in FIGS. 1 and 7encode B. subtilis Dsb1 and Dsb2. The present invention encompassesnovel gram positive microorganism amino acid variants of the B. subtilisDsb1 and Dsb2 amino acid sequences that are at least 80% identical, atleast 90% identical and at least 95% identical to B. subtilis Dsb1 andDsb2 as long as the amino acid sequence variant is able to function in agram-positive microorganism.

The B. subtilis Dsb1 and Dsb2 were discovered upon a FASTA amino acidsearch of translated B. subtilis genomic DNA sequences vs. An E. colidisulfide bond isomerase amino acid sequence (FASTA search Release 1.0,released on Jun. 11, 1997) parameters being Scoring matrix: GenRunData:blosum50 cmp; variable pamfactor used; Gap creation penalty: 12; and Gapextension penalty: 2). Amino acid alignments are shown in FIGS. 2 and 3.The hydrophilicity profile for B. subtilis Dsb1 and Dsb2, shown in FIGS.5 and 6, show potential membrane spanning regions.

II. Expression Systems

The present invention provides expression systems for the enhancedproduction and secretion of desired heterologous or homologous proteinsin gram-positive microorganisms.

a. Coding Sequences

In the present invention, the vector comprises at least one copy ofnucleic acid encoding a gram-positive microorganism Dsb1 and/or Dsb2andpreferably comprises multiple copies. In a preferred embodiment, thegram-positive microorganism is Bacillus. In another preferredembodiment, the gram-positive microorganism is Bacillus subtilis. In apreferred embodiment, polynucleotides which encode B. subtilis Dsb1 andDsb2, or fragments thereof, or fusion proteins or polynucleotide homologsequences that encode amino acid variants of Dsb1 and Dsb2, may be usedto generate recombinant DNA molecules that direct the expression of Dsb1or Dsb2, or amino acid variants thereof, respectively, in gram-positivehost cells. In a preferred embodiment, the host cell belongs to thegenus Bacillus. In another preferred embodiment, the host cell is B.subtilis.

As will be understood by those of skill in the art, it may beadvantageous to produce polynucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particulargram-positive host cell (Murray E et al (1989) Nuc Acids Res 17:477-508)can be selected, for example, to increase the rate of expression or toproduce recombinant RNA transcripts having desirable properties, such asa longer half-life, than transcripts produced from naturally occurringsequence.

Altered gram positive Dsb1 and Dsb2 polynucleotide sequences which maybe used in accordance with the invention include deletions, insertionsor substitutions of different nucleotide residues resulting in apolynucleotide that encodes the same or a functionally equivalent Dsb1and Dsb2 homolog, respectively. As used herein a “deletion” is definedas a change in either nucleotide or amino acid sequence in which one ormore nucleotides or amino acid residues, respectively, are absent.

As used herein an “insertion” or “addition” is that change in anucleotide or amino acid sequence which has resulted in the addition ofone or more nucleotides or amino acid residues, respectively, ascompared to the naturally occurring gram positive Dsb1 and Dsb2.

As used herein “substitution” results from the replacement of one ormore nucleotides or amino acids by different nucleotides or amino acids,respectively.

The encoded protein may also show deletions, insertions or substitutionsof amino acid residues which produce a silent change and result in afunctionally equivalent gram-positive Dsb1 or Dsb2 variant. Deliberateamino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the variant retains theability to modulate secretion. For example, negatively charged aminoacids include aspartic acid and glutamic acid; positively charged aminoacids include lysine and arginine; and amino acids with uncharged polarhead groups having similar hydrophilicity values include leucine,isoleucine, valine; glycine, alanine; asparagine, glutamine; serine,threonine, phenylalanine, and tyrosine.

The Dsb1 and Dsb2 polynucleotides of the present invention may beengineered in order to modify the cloning, processing and/or expressionof the gene product. For example, mutations may be introduced usingtechniques which are well known in the art, eg, site-directedmutagenesis to insert new restriction sites, to alter glycosylationpatterns or to change codon preference, for example.

In one embodiment of the present invention, a Dsb1 and Dsb2polynucleotide may be ligated to a heterologous sequence to encode afusion protein. A fusion protein may also be engineered to contain acleavage site located between the Dsb1 and Dsb2 nucleotide sequence andthe heterologous protein sequence, so that the Dsb1 and Dsb2 protein maybe cleaved and purified away from the heterologous moiety.

b. Vector Sequences

Expression vectors used in expressing the disulfide bond isomerases ofthe present invention in gram-positive microorganisms comprise at leastone promoter associated with a gram-positive Dsb1 and Dsb2, whichpromoter is functional in the host cell. In one embodiment of thepresent invention, the promoter is the wild-type promoter for theselected disulfide bond isomerase and in another embodiment of thepresent invention, the promoter is heterologous to the disulfide bondisomerase, but still functional in the host cell.

Additional promoters associated with heterologous nucleic acid encodingdesired proteins or polypeptides may be introduced via recombinant DNAtechniques. In one embodiment of the present invention, the host cell iscapable of overexpressing a heterologous protein or polypeptide andnucleic acid encoding one or more disulfide bond isomerase(s) is(are)recombinantly introduced. In one preferred embodiment of the presentinvention, nucleic acid encoding Dsb1 or Dsb2 is stably integrated intothe microorganism genome. In another embodiment, the host cell isengineered to overexpress a disulfide bond isomerase of the presentinvention and nucleic acid encoding the heterologous protein orpolypeptide is introduced via recombinant DNA techniques. The presentinvention encompasses gram-positive host cells that are capable ofoverexpressing other disulfide bond isomerase s known to those of skillin the art, including but not limited to, SecA, SecY, SecE or otherdisulfide bond isomerases known to those of skill in the art oridentified in the future.

In a preferred embodiment, the expression vector contains a multiplecloning site cassette which preferably comprises at least onerestriction endonuclease site unique to the vector, to facilitate easeof nucleic acid manipulation. In a preferred embodiment, the vector alsocomprises one or more selectable markers. As used herein, the termselectable marker refers to a gene capable of expression in thegram-positive host which allows for ease of selection of those hostscontaining the vector. Examples of such selectable markers include butare not limited to antibiotics, such as, erythromycin, actinomycin,chloramphenicol and tetracycline.

c. Transformation

In one embodiment of the present invention, nucleic acid encoding atleast one gram-positive disulfide bond isomerase of the presentinvention is introduced into a gram-positive host cell via an expressionvector capable of replicating within the host cell. Suitable replicatingplasmids for Bacillus are described in Molecular Biological Methods forBacillus, Ed. Harwood and Cutting, John Wiley & Sons, 1990, herebyexpressly incorporated by reference; see chapter 3 on plasmids. Suitablereplicating plasmids for B. subtilis are listed on page 92.

In another embodiment, nucleic acid encoding a gram-positivemicro-organism Dsb1 and/or Dsb2 is stably integrated into themicroorganism genome. Preferred gram-positive host cells are from thegenus Bacillus. Another preferred gram-positive host cell is B.subtilis. Several strategies have been described in the literature forthe direct cloning of DNA in Bacillus. Plasmid marker rescuetransformation involves the uptake of a donor plasmid by competent cellscarrying a partially homologous resident plasmid (Contente et al.,Plasmid 2:555-571 (1979); Haima et al., Mol. Gen. Genet. 223:185-191(1990); Weinrauch et al., J. Bacteriol. 154(3):1077-1087 (1983); andWeinrauch et al., J. Bacteriol. 169(3):1205-1211 (1987)). The incomingdonor plasmid recombines with the homologous region of the resident“helper” plasmid in a process that mimics chromosomal transformation.

Transformation by protoplast transformation is described for B. subtilisin Chang and Cohen, (1979) Mol. Gen. Genet 168:111-115; for B.megateriumin Vorobjeva et al., (1980) FEMS Microbiol. Letters 7:261-263; for B.amyloliquefaciens in Smith et al., (1986) Appl. and Env. Microbiol.51:634; for B. thuringiensis in Fisher et al., (1981) Arch. Microbiol.139:213-217; for B. sphaericus in McDonald (1984) J. Gen. Microbiol.130:203; and B.larvae in Bakhiet et al., (1985) 49:577. Mann et al.,(1986, Current Microbiol. 13:131-135) report on transformation ofBacillus protoplasts and Holubova, (1985) Folia Microbiol. 30:97)disclose methods for introducing DNA into protoplasts using DNAcontaining liposomes.

III. Identification of Transformants

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression shouldbe confirmed. For example, if the nucleic acid encoding Dsb1 and Dsb2 isinserted within a marker gene sequence, recombinant cells containing theinsert can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with nucleic acidencoding the disulfide bond isomerase under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the disulfide bond isomeraseas well.

Alternatively, host cells which contain the coding sequence for adisulfide bond isomerase and express the protein may be identified by avariety of procedures known to those of skill in the art. Theseprocedures include, but are not limited to, DNA-DNA or DNA-RNAhybridization and protein bioassay or immunoassay techniques whichinclude membrane-based, solution-based, or chip-based technologies forthe detection and/or quantification of the nucleic acid or protein.

The presence of the Dsb1 or Dsb2 polynucleotide sequence can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes,portions or fragments derived from the B. subtilis Dsb1 or Dsb2polynucleotide.

IV. Secretion Assays

Means for determining the levels of secretion of a heterologous orhomologous protein in a gram-positive host cell and detecting secretedproteins include, using either polyclonal or monoclonal antibodiesspecific for the protein. Examples include enzyme-linked immunosorbentassay (ELISA), radioimmunoassay (RIA) and fluorescent activated cellsorting (FACS). These and other assays are described, among otherplaces, in Hampton R et al (1990, Serological Methods a LaboratoryManual. APS Press, St Paul Minn.) and Maddox Del. et al (1983, J Exp Med158:1211).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic and amino acidassays. Means for producing labeled hybridization or PCR probes fordetecting specific polynucleotide sequences include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the nucleotide sequence, or any portion ofit, may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3 or SP6 and labeled nucleotides.

A number of companies such as Pharmacia Biotech (Piscataway N.J.),Promega (Madison Wis.), and US Biochemical Corp (Cleveland Ohio) supplycommercial kits and protocols for these procedures. Suitable reportermolecules or labels include those radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles and the like. Patents teachingthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also,recombinant immunoglobulins may be produced as shown in U.S. Pat. No.4,816,567 and incorporated herein by reference.

V. Purification of Proteins

Gram positive host cells transformed with polynucleotide sequencesencoding heterologous or homologous protein may be cultured underconditions suitable for the expression and recovery of the encodedprotein from cell culture. The protein produced by a recombinantgram-positive host cell comprising a disulfide bond isomerase of thepresent invention will be secreted into the culture media. Otherrecombinant constructions may join the heterologous or homologouspolynucleotide sequences to nucleotide sequence encoding a polypeptidedomain which will facilitate purification of soluble proteins (Kroll DJet al (1993) DNA Cell Biol 12:441-53).

Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals (Porath J (1992) Protein Expr Purif3:263-281), protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). The inclusion of acleavable linker sequence such as Factor XA or enterokinase (Invitrogen,San Diego Calif.) between the purification domain and the heterologousprotein can be used to facilitate purification.

VI. Uses of the Present Invention

Genetically Engineered Host Cells

The present invention provides genetically engineered host cellscomprising nucleic acid encoding gram-positive microorganism Dsb1 and/orDsb2. In one preferred embodiment, the nucleic acid is stably integratedinto the microorganism. In another preferred embodiment, the Dsb1 and/orDsb2 have the nucleic acid sequence shown in FIGS. 1 and 7,respectively. The host cell may comprise nucleic acid encodingadditional secretions factors.

The preferred embodiment, the host cell is genetically engineered toproduce a desired protein or polypeptide. In a preferred embodiment, thehost cell is a Bacillus. In another preferred embodiment, the host cellis Bacillus subtilis.

In an alternative embodiment, a host cell is genetically engineered toproduce a gram-positive Dsb1 or Dsb2.

Polynucleotides

B. subtilis Dsb1 or Dsb2 polynucleotides, or any part thereof, providesthe basis for detecting the presence of gram-positive microorganismpolynucleotide homologs through hybridization techniques and PCRtechnology.

Accordingly, one aspect of the present invention is to provide fornucleic acid hybridization and PCR probes derived from B. subtilis Dsb1or Dsb2 which can be used to detect other gram positive microorganismpolynucleotide sequences, including genomic and cDNA sequences.

EXAMPLE I

Preparation of a Genomic library

The following example illustrates the preparation of a Bacillus genomiclibrary.

Genomic DNA from Bacillus cells is prepared as taught in CurrentProtocols In Molecular Biology vol. 1, edited by Ausubel et al., JohnWiley & Sons, Inc. 1987, chapter 2. 4.1. Generally, Bacillus cells froma saturated liquid culture are lysed and the proteins removed bydigestion with proteinase K. Cell wall debris, polysaccharides, andremaining proteins are removed by selective precipitation with CTAB, andhigh molecular weight genomic DNA is recovered from the resultingsupernatant by isopropanol precipitation. If exceptionally clean genomicDNA is desired, an additional step of purifying the Bacillus genomic DNAon a cesium chloride gradient is added.

After obtaining purified genomic DNA, the DNA is subjected to Sau3Adigestion. Sau3A recognizes the 4 base pair site GATC and generatesfragments compatible with several convenient phage lambda and cosmidvectors. The DNA is subjected to partial digestion to increase thechance of obtaining random fragments.

The partially digested Bacillus genomic DNA is subjected to sizefractionation on a 1% agarose gel prior to cloning into a vector.Alternatively, size fractionation on a sucrose gradient can be used. Thegenomic DNA obtained from the size fractionation step is purified awayfrom the agarose and ligated into a cloning vector appropriate for usein a host cell and transformed into the host cell.

EXAMPLE II

Detection of Gram-positive Microorganism Disulfide Bond Isomerase

The following example describes the detection of gram-positivemicroorganism Dsb1 or Dsb2.

DNA derived from a gram-positive microorganism is prepared according tothe methods disclosed in Current Protocols in Molecular Biology, Chap. 2or 3. The nucleic acid is subjected to hybridization and/or PCRamplification with a probe or primer derived from Dsb1 or Dsb2.

The nucleic acid probe is labeled by combining 50 pmol of the nucleicacid and 250 mCi of [gamma ³²P] adenosine triphosphate (Amersham,Chicago Ill.) and T4 polynucleotide kinase (DuPont NEN®, Boston Mass.).The labeled probe is purified with Sephadex G-25 super fine resin column(Pharmacia). A portion containing 10⁷ counts per minute of each is usedin a typical membrane based hybridization analysis of nucleic acidsample of either genomic or cDNA origin.

The DNA sample which has been subjected to restriction endonucleasedigestion is fractionated on a 0.7 percent agarose gel and transferredto nylon membranes (Nytran Plus, Schleicher & Schuell, Durham N.H.).Hybridization is carried out for 16 hours at 40 degrees C. To removenonspecific signals, blots are sequentially washed at room temperatureunder increasingly stringent conditions up to 0.1×saline sodium citrateand 0.5% sodium dodecyl sulfate. The blots are exposed to film forseveral hours, the film developed and hybridization patterns arecompared visually to detect polynucleotide homologs of B. subtilis Dsb1or Dsb2. The homologs are subjected to confirmatory nucleic acidsequencing. Methods for nucleic acid sequencing are well known in theart. Conventional enzymatic methods employ DNA polymerase Klenowfragment, SEQUENASE®) (US Biochemical Corp, Cleveland, Ohio) or Taqpolymerase to extend DNA chains from an oligonucleotide primer annealedto the DNA template of interest.

Various other examples and modifications of the foregoing descriptionand examples will be apparent to a person skilled in the art afterreading the disclosure without departing from the spirit and scope ofthe invention, and it is intended that all such examples ormodifications be included within the scope of the appended claims. Allpublications and patents referenced herein are hereby incorporated byreference in their entirety.

5 1 444 DNA Bacillus subtilis 1 atgaatacaa gatatgtaaa atcattttttttattactgt tttttctctc tttctttggc 60 acaatggcta gtttattcta cagtgagatcatgcatttca aaccatgtgt tctatgttgg 120 tatcaaagaa tatttctata tcctatacctattatcttac taataggctt attaaaaaaa 180 gatcttaatt cgatatttta tgttgttttcctttcatcaa ttggattgat tattgcgttt 240 tatcattata ttatccaact tacacaaagcaaaagtgtcg tatgtgaaat tggaaccaac 300 agctgcgcaa aaattgaagt agagtatctaggctttatta cattaccctt aatgagttca 360 gtatgttttg cattgatatt tggtataggactgaaattaa ttatcaaaag caagaaatta 420 aaacaaaatc aacatgtata taat 444 2148 PRT Bacillus subtilis 2 Met Asn Thr Arg Tyr Val Lys Ser Phe Phe LeuLeu Leu Phe Phe Leu 1 5 10 15 Ser Phe Phe Gly Thr Met Ala Ser Leu PheTyr Ser Glu Ile Met His 20 25 30 Phe Lys Pro Cys Val Leu Cys Trp Tyr GlnArg Ile Phe Leu Tyr Pro 35 40 45 Ile Pro Ile Ile Leu Leu Ile Gly Leu LeuLys Lys Asp Leu Asn Ser 50 55 60 Ile Phe Tyr Val Val Phe Leu Ser Ser IleGly Leu Ile Ile Ala Phe 65 70 75 80 Tyr His Tyr Ile Ile Gln Leu Thr GlnSer Lys Ser Val Val Cys Glu 85 90 95 Ile Gly Thr Asn Ser Cys Ala Lys IleGlu Val Glu Tyr Leu Gly Phe 100 105 110 Ile Thr Leu Pro Leu Met Ser SerVal Cys Phe Ala Leu Ile Phe Gly 115 120 125 Ile Gly Leu Lys Leu Ile IleLys Ser Lys Lys Leu Lys Gln Asn Gln 130 135 140 His Val Tyr Asn 145 3176 PRT Bacillus subtilis 3 Met Leu Arg Phe Leu Asn Gln Cys Ser Gln GlyArg Gly Ala Trp Leu 1 5 10 15 Leu Met Ala Phe Thr Ala Leu Ala Leu GluLeu Thr Ala Leu Trp Phe 20 25 30 Gln His Val Met Leu Leu Lys Pro Cys ValLeu Cys Ile Tyr Glu Arg 35 40 45 Cys Ala Leu Phe Gly Val Leu Gly Ala AlaLeu Ile Gly Ala Ile Ala 50 55 60 Pro Lys Thr Pro Leu Arg Tyr Val Ala MetVal Ile Trp Leu Tyr Ser 65 70 75 80 Ala Phe Arg Gly Val Gln Leu Thr TyrGlu His Thr Met Leu Gln Leu 85 90 95 Tyr Pro Ser Pro Phe Ala Thr Cys AspPhe Met Val Arg Phe Pro Glu 100 105 110 Trp Leu Pro Leu Asp Lys Trp ValPro Gln Val Phe Val Ala Ser Gly 115 120 125 Asp Cys Ala Glu Arg Gln TrpAsp Phe Leu Gly Leu Glu Met Pro Gln 130 135 140 Trp Leu Leu Gly Ile PheIle Ala Tyr Leu Ile Val Ala Val Leu Val 145 150 155 160 Val Ile Ser GlnPro Phe Lys Ala Lys Lys Arg Asp Leu Phe Gly Arg 165 170 175 4 414 DNABacillus subtilis 4 atgaaaaata gaatcgtatt tttatatgct tcctgggttgtggctcttat cgctatgctg 60 ggcagcctgt atttcagtga aatcagaaag tttattccatgtgaactgtg ctggtaccag 120 cgtatcctca tgtatccgct cgtcctgatt ttaggcatcgccacctttca aggggacaca 180 cgagtgaaaa aatatgtgct cccgatggcg attattggggcattcatttc gatcatgcat 240 tacttagagc aaaaagtgcc cggctttagc ggcattaagccatgtgtcag cggcgtgccg 300 tgctcgggcc aatatattaa ctggtttggt tttattacgattccattcct ggccctgatt 360 gcttttatcc tgattatcat ttttatgtgc ctgctgaaaggcgaaaaatc tgaa 414 5 138 PRT Bacillus subtilis 5 Met Lys Asn Arg IleVal Phe Leu Tyr Ala Ser Trp Val Val Ala Leu 1 5 10 15 Ile Ala Met LeuGly Ser Leu Tyr Phe Ser Glu Ile Arg Lys Phe Ile 20 25 30 Pro Cys Glu LeuCys Trp Tyr Gln Arg Ile Leu Met Tyr Pro Leu Val 35 40 45 Leu Ile Leu GlyIle Ala Thr Phe Gln Gly Asp Thr Arg Val Lys Lys 50 55 60 Tyr Val Leu ProMet Ala Ile Ile Gly Ala Phe Ile Ser Ile Met His 65 70 75 80 Tyr Leu GluGln Lys Val Pro Gly Phe Ser Gly Ile Lys Pro Cys Val 85 90 95 Ser Gly ValPro Cys Ser Gly Gln Tyr Ile Asn Trp Phe Gly Phe Ile 100 105 110 Thr IlePro Phe Leu Ala Leu Ile Ala Phe Ile Leu Ile Ile Ile Phe 115 120 125 MetCys Leu Leu Lys Gly Glu Lys Ser Glu 130 135

What is claimed is:
 1. An expression vector comprising a nucleic acidencoding a disulfide bond isomerase (Dsb), wherein said nucleic acidsequence is under the control of an expression signal capable ofexpressing said Dsb in a gram-positive microorganism, wherein said Dsbhas an amino acid sequence selected from the group consisting of: a) SEQID NO:2, b) SEQ ID NO:5, c) an amino acid sequence that is at least 95%identical to SEQ ID NO:2, and d) an amino acid sequence that is at least95% identical to SEQ ID NO:5.
 2. The expression vector of claim 1wherein the gram-positive microorganism is a Bacillus.
 3. An expressionvector comprising a nucleic acid sequence encoding a disulfide bondisomerase (Dsb) having an amino acid sequence selected form the groupconsisting of SEQ ID NO:2 and SEQ ID NO:5, wherein said Dsb is under thecontrol of an expression signal capable of expressing said Dsb in aBacillus host cell.
 4. The expression vector of claim 3 wherein thenucleic acid sequence encodes the Dsb having the amino acid sequence ofSEQ ID NO:2.
 5. The expression vector of claim 3 wherein the nucleicacid sequence encodes the Dsb having the amino acid sequence of SEQ IDNO:5.
 6. The expression vector of claim 3 wherein the nucleic acidsequence comprises the sequence shown in SEQ ID NO:1.
 7. The expressionvector of claim 3 wherein the nucleic acid sequence comprises thesequence shown in SEQ ID NO:
 4. 8. A gram positive host cell comprisingthe expression vector of claim
 6. 9. A Bacillus host cell comprising theexpression vector of claim
 3. 10. The host cell of claim 9 wherein theBacillus is selected from the group consisting of B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alcalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus and B. thuringiensis.
 11. The host cell of claim 9 wherein saidhost cell is capable of expressing a heterologous protein.
 12. The hostcell of claim 11 wherein said heterologous protein is selected from thegroup consisting of hormones, enzymes, growth factor and cytokines. 13.The host cell of claim 12 wherein said heterologous protein is anenzyme.
 14. The host cell of claim 13 wherein said enzyme is selectedfrom the group consisting of a proteases, amylases, cellulases,xylanases, reductases and lipases, oxidases, esterases, isomerases,kinases and phosphatases.
 15. The host cell of claim 14 wherein saidenzyme is a protease, an amylase, a cellulase or a lipase.
 16. The hostcell of claim 10 wherein the Bacillus is a B. subtilis.
 17. The Bacillussubtilis host cell of claim 16 wherein the host cell further comprises aheterologous protein.
 18. The Bacillus subtilis host cell of claim 17wherein the heterologous protein is an enzyme.
 19. A method forsecreting a protein in a gram-positive microorganism comprising thesteps of: a) transforming a gram-positive microorganism with anexpression vector according to claim 1, said gram-positive microorganismfurther comprising a nucleic acid sequence encoding a homologous orheterologous protein; b) culturing said microorganism under conditionssuitable for expression of said Dsb; and c) allowing expression andsecretion of said homologous or heterologous protein.
 20. The method ofclaim 19 wherein said protein is homologous to said host cell.
 21. Themethod of claim 19 wherein said protein is heterologous to said hostcell.
 22. The method of claim 21 wherein said heterologous protein isselected from the group consisting of hormones, enzymes, growth factorsand cytokines.
 23. The method of claim 22 wherein said heterologousprotein is an enzyme.
 24. The method of claim 23 wherein said enzyme isselected from the group consisting of proteases, amylases, cellulases,xylanases, reductases, lipases, oxidases, esterases, isomerases, kinasesand phosphatases.
 25. The method of claim 19 wherein said gram-positiveorganism Microorganism is a Bacillus.
 26. The method of claim 25 whereinsaid Bacillus is selected from the group consisting of B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alcalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus and B. thuringiensis.
 27. The method of claim 14, wherein therecombinantly introduced protein is and enzyme.
 28. The method of claim27, wherein the recombinantly introduced enzyme is a protease, anamylase, a cellulase or a lipase.